Arka Acharyya, Ramakrishna Mission Vivekananda Centenary College, Rahara
Researcher Jun Hee Lee and his team at the University of Michigan developed a new technique to visualize using high-throughput sequencing instead of a microscope. They named it technology Sequence-Scope technology or Seq-Scope. The development of light and electron microscopes has a profound role in the study of histology, the branch of biology that deals with microscopic studies of tissue anatomy. Immunohistochemistry and RNA-in-situ hybridization are two conventional techniques that detect proteins and mRNAs from the histological slides. These techniques make it easy to understand various pathological and physiological processes and capable of diagnosis various types of disease at a molecular level. But, at a time, standard immunohistochemistry and RNA-in-situ hybridization are not able to identify more than one target molecular species. Limited information is obtained from a single experimental session. To overcome this limitation, the technique of sequence-scope technology has been developed. Sequence-scope technology is a new method by which all types of gene expressions are visualized from a tissue sample without a microscope. The technique is capable of visualization every gene expression as well as single cells and their structures at incredibly high resolution. Researchers claim that this advanced technology can detect 0.6 micrometers or 66 times smaller than a human hair.
According to Jun Lee, Sequence-scope technology is an advanced barcoding method with a resolution nearly comparable to an optical microscope. Technology is based on a solid phase amplification that randomly barcoded signal molecule oligonucleotides sequences – the pattern of A, T, G, and C. Using these barcodes, a computer can able to identify every gene within a tissue sample. This information builds a Google-like database of all mRNAs transcribed from the genome. An illumine sequencing-by-synthesis platform is used in it. Researchers explain that the Sequence-scope technology experiments are split up into two rounds of sequencing steps: first-sequence and second-sequence. First-sequence produces a physical array of barcoded RNA-capture molecules and a unique map of barcodes, called HDMI. HDMI is a high-definition map coordinate identifier, a spatial barcode form with 20-32 random nucleotide sequences.
A restriction enzyme named Oligo-dT is used for the first sequence. The enzyme has a unique RNA capturing domain. Second-sequence captures mRNAs that come from tissue and that placed on a physical array from the first sequence. The captured molecules contain both barcode information and cDNA. Researchers explain that for each high definition map coordinate identifier, the first sequence provides unique coordinate information. On the other hand, the second sequence provides information about captured cDNA. Then a spatial digital gene expression (DGE) matrix is formed by combining the first and second sequences. The DGE matrix is crucial for various analyses, including clustering assays and gene expression visualization.
Sequence-scope technology is fast, straightforward, and easy to implement. The technology is capable to generate up to 150 HDMIs in a 100-micrometer square area. This resolution is sufficient for analyzing various cellular components including single cell types, subtypes, and also sub-cellular organizations of nucleus, cytoplasm, and mitochondria. From a tissue section, Sequence-Scope visualizes transcriptomic architectures at multiple histological scales. These include tissue zonation of crypt-surface (colon), portal-central (liver), and inflammation fibrosis (injured liver) axes. Researchers demonstrate the potentiality of the technique using normal and injured liver cells. They successfully identified dying liver cells surrounded with inflamed immune cells and live cells with altered gene expression. Jun Lee says that the technique can produce an unbiased systemic process to analyze genes. With a combination of other single-cell RNA sequencing techniques, it can accelerate scientific discoveries. It may lead to a new paradigm in molecular diagnosis, Jun Lee also adds.
Also read: Newly discovered glial cells can revolutionize brain repair
Reference:
- Cho, Chun-Seok, et al. “Microscopic Examination of Spatial Transcriptome Using Seq-Scope.” Cell, June 2021. DOI: https://doi.org/10.1016/j.cell.2021.05.010
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